10.3: Details of Transcription

You can find a well-written summary of transcription in prokaryotes and eukaryotes on the National Institutes of Health (NIH) website at Transcription in Prokaryotes and Eukaryotes. At the link as well as here in this book, you will encounter proteins that bind DNA. Some proteins bind DNA to regulate transcription, inducing or silencing the transcription of a gene; we will discuss their role in the regulation of gene expression later. Other proteins interact with DNA simply to allow transcription; these include one or more that, along with RNA polymerase itself, must bind to the gene promoter to initiate transcription. We will look at bacterial transcription first.

10.3.1. Details of Transcription in Prokaryotes

In E. coli, a single RNA polymerase transcribes all kinds of RNA, associating with one of several sigma factor proteins (\(\sigma\)−factors) to initiate transcription. Different promoter sequences and their corresponding \(\sigma\)−factors play roles in the transcription of different genes (Figure. 10.7).

Figure 10.7: All transcription begins when RNA polymerase binds to a two-part 'promoter' in mRNA (here shown as the '-35' and 'pribnow box' sequences). Bacterial RNA polymerase is associated with 'sigma factors' that allow it to recognize and bind to the promoter. Different 'sigma factors' recognize different promoter sequences.

In the absence of a \(\sigma\)−factor, E. coli RNA polymerase transcribes RNA, but it does so at a high rate and from random sequences in the chromosome. With a \(\sigma\)−factor bound to the RNA polymerase, the complex seemingly scans the DNA and recognizes and binds to the promoter sequence of a gene. The overall transcription rate is slower, but rather than random bits of the bacterial genome, only genes are transcribed! The Pribnow box, named for its discoverer, was the first RNA polymerase-binding sequence in a promoter to be characterized.

One way that bacteria regulate which genes are expressed is to selectively control the cellular concentrations of different \(\sigma\)−factors available to the Pribnow box. \(\sigma^{70}\) is the main \(\sigma\)−factor of bacterial transcription initiation \(\sigma^{54}\), a structurally unrelated factor regulates a variety of different genes, typically in response to stress (e.g., high temperature or antibiotic attack). Still other “alternative” \(\sigma\)−factors participate in activating specific genes or gene subsets (Sigma Subunits and Bacterial Regulation). We shall see more modes of prokaryotic gene regulation in the next chapter.

Typically, the \(\sigma\)−factor falls off the RNA polymerase very soon after transcription is initiated, which then continues unwinding the double helix and elongating the transcript (Figure 10.8).

Figure 10.8: After a bacterial RNA polymerase binds to the promoter (at the left), translation begins. No longer needed to initiate transcription, the 'sigma factor' dissociates from the polymerase (at the right), which then continues transcribing the RNA.

Elongation is the successive addition of nucleotides complementary to their DNA templates by forming phosphodiester linkages between nucleotides. The enzymatic condensation reactions of elongation are much like DNA polymerase-catalyzed elongation during replication.

CHALLENGE

How is chain (strand) elongation during replication different from chain elongation during transcription? Try to describe the difference in your own words.

There are two ways that bacterial RNA polymerase “knows” when it has reached the end of a transcription unit. In one case, as the RNA polymerase nears the 3’ end of the nascent transcript, it transcribes a 72-base, C-rich region. At this point, a termination factor called the rho protein binds to the nascent RNA strand; rho is an ATP-dependent helicase that breaks the H-bonds between the RNA and the template DNA strand, thus preventing further transcription. Figure 10.9 (below) illustrates rho-dependent termination.

Figure 10.9: Rho-dependent termination is one mechanism by which an RNA polymerase knows when it has finished transcribing. The Rho protein is an RNA-DNA helicase that recognizes a sequence near the end of the gene and transcript that unwinds the nascent RNA from the gene, ending transcription.

In the other mechanism of termination, a sequence near the 3’ end of the transcript folds into a hairpin loop secondary structure, which serves as a termination signal, also causing dissociation of the RNA polymerase, template DNA, and the new RNA transcript. The role of the hairpin loop in rho-independent termination is shown in Figure 10.10.

Figure 10.10: Rho-independent termination is another mechanism for terminating bacterial transcription. A sequence near the end of the nascent transcript has internal complementary bases that fold into a hairpin-loop that acts as a termination signal.

191 Details of Prokaryotic Transcription

10.3.2. Details of Transcription in Eukaryotes

Unlike prokaryotes, eukaryotes use three different RNA polymerases to synthesize the three major RNAs (Figure 10.11).

Figure 10.11: The work of Eukaryotic transcription is more complex than that of prokaryotes. The latter transcribe all RNAs with a single RNA polymerase. Eukaryotes catalyze transcription of their rRNAs, mRNAs and tRNAs with different RNA polymerases.

With the help of initiation proteins, each RNA polymerase forms an initiation complex by combining with several transcription factors (TFs). Once the DNA at the start site of transcription unwinds, RNA polymerases catalyze the successive formation of phosphodiester linkages to elongate the transcript. Note that catalysis of the synthesis of most of the RNA in a eukaryotic cell (i.e., rRNAs) is by RNA polymerase I!

These condensation reactions add ribose nucleotides to the free 3’ end of a growing RNA molecule in reactions that are like those that elongate DNA strands. Unfortunately, the details of the eukaryotic transcription termination are not as well understood as they are in bacteria. Here we focus on initiation, followed by discussion of the processing of different eukaryotic RNAs into ready-to-use molecules.

10.3.2.a Eukaryotic mRNA Transcription

Transcription of eukaryotic mRNAs by RNA polymerase II begins with sequential assembly of a eukaryotic initiation complex at a gene promoter. The typical promoter for a protein-encoding gene contains a -T-A-T-A- sequence motif, or TATA box, and other short upstream sequences that recruit components of the initiation complex. The many steps of eukaryotic mRNA transcription initiation are illustrated in Figure 10.12 (below).

Figure 10.12: Eukaryotic mRNA-transcription initiation starts with recognition of a promoter TATA box by initiation factor TFIID (TBP) (1). The association of a series of initiation factors follows (2, 3). RNA polymerase II binds last, and its phosphorylation starts mRNA transcription using the UTP, ATP, CTP, and GTP nucleotide precursors (4).

TATA-binding protein (TBP) first binds to the TATA box, along with TFIID (transcription factor IID). This intermediate in turn recruits TFIIA and TFIIB. Then TFIIE, TFIIF, and TFIIH are added to form the core initiation complex. Finally, several other initiation factors and RNA polymerase II bind to complete the mRNA transcription-initiation complex. Phosphorylation adds several phosphates to the amino terminus of the RNA polymerase, after which some of the transcription factors dissociate from the initiation complex. The remaining RNA-polymerase transcription-factor complex can now start making the mRNA.

The first task of the complete initiation complex is to unwind the template DNA strands at the start site of transcription. Unlike prokaryotic RNA polymerase, eukaryotic RNA Polymerase II does not have an inherent DNA helicase activity. For this, eukaryotic gene transcription relies on the multi-subunit TFIIH protein, two of whose subunits have an ATP-dependent helicase activity.

Once transcription is initiated, helicase activity is no longer required for continued elongation of the RNA strand. Consistent with the closer relationship of archaea to eukaryotes (rather than to prokaryotes), archaeal mRNA transcription initiation more closely resembles transcription in eukaryotes, albeit requiring fewer initiation factors during formation of an initiation complex.

192-2 Eukaryotic mRNA Transcription

A significant difference between prokaryotic and eukaryotic transcription is that RNA polymerases and other proteins involved at a eukaryotic gene promoter do not see naked DNA. Instead, they recognize specific DNA sequences behind a coat of chromatin proteins, and some of whose bases have been chemically modified.

On the other hand, all proteins that interact with DNA have a common a need to recognize the DNA sequences to which they must bind—within the double helix. In other words, they must see the bases in the interior of the helix, not its uniformly electronegative phosphodiester backbone surface. To this end, they must penetrate the DNA, usually through the major groove of the double helix. We will see that DNA regulatory proteins face the same problems in achieving specific shape-based interactions!

193-2 Recognition of Transcription Factors at Promoters

10.3.2.b Eukaryotic tRNA and 5S rRNA Transcription

It must have been something of a surprise to discover that the promoter sequence that binds RNA polymerase III to start transcribing 5S rRNAs and tRNAs by is not upstream of the transcribed sequence; rather, it lies within the transcribed sequence. To begin this unusual process, RNA polymerase III associates with structural relatives of the RNA polymerase II transcription factors (TFs) to form a core initiation complex at these “internal promoters”.

After additional, gene-specific TFs combine with this core complex, the finished initiation complex repositions RNA polymerase III from the internal promoter sequence to the upstream transcription start site to begin RNA synthesis. As a result, the final 5S rRNA and tRNA transcripts still contain retain the ‘promoter’ sequence! Note that, unlike initiation of mRNA transcription, RNA polymerase III-dependent transcription initiation does not require an independent DNA unwinding activity (i.e., a helicase). Figure 10.13 (below) illustrates the transcription of a 5S rRNA (or a tRNA) by RNA polymerase III.

Figure 10.13: RNA polymerase III initiates 5S rRNA and tRNA transcription from an internal promoter. After binding to the promoter, the polymerase repositions itself near the transcription start site (1). Transcription begins, producing 5S rRNAs and tRNAs that include their promoter sequences (2).

10.3.2.c Transcription of the Other Eukaryotic rRNAs

The production of the other rRNAs requires RNA polymerase I. A 45S precursor rRNA (pre-rRNA) is made and then processed into 28S, 18S, and 5.8S rRNAs (see section 10.5). RNA polymerase I and “core” TFs form a core initiation complex at the upstream promoters of the 45S genes. The addition of several gene–specific TFs completes assembly of the initiation complex, which then unwinds the promoter DNA, again without a separate helicase.

More information about the structure and function of eukaryotic transcription initiation complexes can be had at Three Transcription Initiation Complexes. As already noted, transcription termination is not as well understood in eukaryotes as in prokaryotes. Coupled termination and polyadenylation steps common to most prokaryotic mRNAs are discussed in more detail in the next section, and a useful summary can be found at the NIH-NCBI website, Eukaryotic Transcription Termination.